Project Summary/ Abstract Neural tube closure defects and congenital heart defects are common developmental disorders, affecting 1 in 1000 and 1 in 100 live births, respectively. During embryonic development, cells must be able to sense direction and coordinate collective behaviors to avoid these severe malformations. Although the signaling pathway that imparts directionality between neighboring cells is known, uncovering the mechanisms that transmit this information on the order of thousands of cells has stalled. Fortuitously, spontaneous mutations in small mammals have given rise to animals that are prized for region specific disruptions in fur orientation. I hypothesize that the mutations disrupt long-range directional cues. The objective of this proposal is to utilize spontaneous mutations to identify long-range directional cues in the mammalian epidermis. This work will shed light on the etiology of complex congenital diseases, which will assist in the development of in utero therapies and the generation of artificial organs. A key molecular pathway that allows cells to communicate directional information is the Planar Cell Polarity (PCP) pathway, without which severe developmental defects arise. The core pathway consists of three transmembrane proteins that form asymmetric complexes that are localized to opposite sides of a cell. Through both positive extracellular feedback and negative intracellular feedback, this asymmetry is propagated locally but the mechanism that orients asymmetry across entire tissues is poorly understood. Although the core pathway components were identified through forward genetic screens in Drosophila, this method has been less successful in uncovering global polarity cues. The mouse skin provides an ideal system to identify long-range polarity cues. The expansive tissue is decorated with thousands of hair follicles (HFs) that are all oriented in the same direction through a PCP driven process. Remarkably, we showed the back skin is compartmentalized into regional domains that influence the direction of PCP signaling. This data suggests that region specific long-range cues cooperate to coordinate PCP and HF orientation across the entire tissue. In support of this idea, spontaneous mutations in small mammals produce animals with distinct, region-specific disruptions in hair follicle orientation. To investigate whether these mutations affect long-range polarity genes, I will identify the causative mutations through genomic sequencing and characterize the effect the mutations have on tissue polarity using genetics and image analysis. I will then interrogate the ability of the identified proteins to act as long-range polarity cues in vivo and in organotypic skin culture. Upon completion of this study, we will identify and understand new mechanisms that coordinate collective behaviors across great distances during tissue morphogenesis.